Study of key residues in MERS-CoV and SARS-CoV-2 main proteases for resistance against clinically applied inhibitors nirmatrelvir and ensitrelvir

The Middle East Respiratory Syndrome Coronavirus (MERS-CoV) is an epidemic, zoonotically emerging pathogen initially reported in Saudi Arabia in 2012. MERS-CoV has the potential to mutate or recombine with other coronaviruses, thus acquiring the ability to efficiently spread among humans and become pandemic. Its high mortality rate of up to 35% and the absence of effective targeted therapies call for the development of antiviral drugs for this pathogen. Since the beginning of the SARS-CoV-2 pandemic, extensive research has focused on identifying protease inhibitors for the treatment of SARS-CoV-2. Our intention was therefore to assess whether these protease inhibitors are viable options for combating MERS-CoV. To that end, we used previously established protease assays to quantify inhibition of SARS-CoV-2, MERS-CoV and other main proteases. Nirmatrelvir inhibited several of these proteases, whereas ensitrelvir was less broadly active. To simulate nirmatrelvir’s clinical use against MERS-CoV and subsequent resistance development, we applied a safe, surrogate virus-based system. Using the surrogate virus, we previously selected hallmark mutations of SARS-CoV-2-Mpro, such as T21I, M49L, S144A, E166A/K/V and L167F. In the current study, we selected a pool of MERS-CoV-Mpro mutants, characterized the resistance and modelled the steric effect of catalytic site mutants S142G, S142R, S147Y and A171S.


Files 1 to 3
File 1: Sequence identity distances table of alignment 5.
File 2: Sequence identity distances table of alignment 6.
File 3: Sequence identity distances table of alignment 7.

Table 1 .
Superposition matrix using residues 9 -194 of chains A (inhibitor binding domain) for M pro structures from different viruses indicated by their PDB ID.Secondary Structure Matching (SSM) superposition on Cα backbone atoms, RMSD in Å.As expected, global atomic RMSD differences between apo and inhibitor-bound forms of the same virus are generally smaller than differences between structures of distant viruses.The differences in inhibitor binding require a detailed local analysis of binding pocket conformation and ligand poses based on experimental structures or virtual docking results.

Table 2 .
Overview of all substitutions generated during selection experiments with chimeric VSV-M pro variants.GISAID: Global Initiative on Sharing All Influenza Data, reference data bank with most SARS-CoV-2 sequence depositions.